The invention relates to a T7 expression system, method for its production and use thereof for antibiotic-free production of recombinant proteins.
T7 expression systems are used for the expression of recombinant proteins. As a rule the T7 promoter sequence is located on a vector and the coding region for a recombinant protein downstream therefrom after a ribosome binding site. Since no T7 polymerase is produced by the expression organism, such an expression system is only functional when the T7 polymerase has been artificially introduced into the system. The commonest method for this is the genomic anchoring of a T7 expression cassette in the production organism. A T7 polymerase-based prokaryotic system for the expression of proteins in BL21 (DE3) Escherichia coli cells has long been known. In this system, the integration of the T7 polymerase into the E. coli genome takes place with the aid of the DE3 lambda phage into the lambda attachment site of E. coli. In the system, the T7 phage polymerase recognizes the T7 phage promoter. However, this system has the disadvantage that the plasmid stability during target gene expression is low. Numerous studies have attempted to stabilize the expression level of the T7 expression system, but so far without appreciable success. Moreover, E. coli contains ca. 40 kb lambda phage DNA. This phage DNA is undesired, at least in systems for the expression of pharmaceutical proteins. Furthermore, in T7 expression systems expression vectors which contain an antibiotic resistance gene as selection marker for the cloning and selection during the expression have always hitherto been used.
However, the use of antibiotics for the selection of plasmids is a critical disadvantage, particularly in the production of therapeutic proteins. An antibiotic-free method is more acceptable to the regulatory authorities, since the product will be safer for the patients and because of the minor end product analysis (elimination of the antibiotic in the product) a markedly more economical process can be constructed. An expression vector which enables a method for antibiotic-free production of proteins is for example known from EP1697523B1. However, this expression vector is not suitable for a T7 expression system.